Approximately 10,000 years ago, a river-dwelling tetra, Astyanax mexicanus invaded multiple caves in the Sierra de El Abra region of Central Mexico. These isolated cave populations underwent rapid evolution of morphological traits such as increased numbers of taste buds and teeth, and the loss of pigmentation and eyes. The constructive evolution of morphological traits can be associated with an adaptive advantage to the cave environment (e.g. increased chemical senses in response to low food supply). The regressive evolution of traits (e.g. loss of pigmentation) is poorly understood at the evolutionary, genetic and molecular levels. Understanding the genetic architecture of regressive evolution by studying the loss of pigmentation in Astyanax cave populations is the primary goal of this research proposal. How many genes are involved in evolution of pigmentation loss? When this trait evolves in parallel in different populations (different caves) are the same genetic pathways utilized to achieve the trait? Are regressive genetic changes structural or regulatory? These questions can be addressed by understanding pigmentation loss in Astyanax mexicanus cave-adapted populations. Our previous QTL analyses and mutation screening implicate both Mc1r and Oca2 as important pigmentation genes in Astyanax. Deeper analysis into the regulation of Mc1r and Oca2 as well as their integrity in additional cave populations will allow for broader evolutionary insights into the nature of regressive evolution. Aims 1 and 2 will focus on determining the minimal, necessary regulatory sequences for both the Mc1r and Oca2 genes in surface Astyanax mexicanus. These sequences will then be screened for mutations in cave fish populations. Aim 3 will more broadly address which genes and what types of genetic lesions lead to regressive evolution of pigmentation traits. A new genetic cross will be performed with surface and cave fish to screen for dominant mutations in the cave fish populations. Identification of dominant mutations leading to the regression of traits will lend valuable insight in to the loss of morphological traits. The questions addressed in this proposal are directly relevant to human pigmentation defects such as albinism (e.g. OMIM 203200) or red hair/fair skin patients with increased risk for melanoma (OMIM 155555). Regulatory mutations and dominant mutations identified from this proposal will provide sequences that could be screened in human patients, for whom no mutations have yet been identified.